The present invention relates generally to the field of synthesizing, analyzing or otherwise processing molecules such as biologically active molecules or polymers. More particularly, the invention relates to a technique for recapturing or recycling biomolecule reagents including nucleotides or nucleic acids in such processes as synthesis, ligation, sequencing, and so forth.
Nucleotides and nucleic acids are processed for many purposes. Nucleic acids may be used, for example, in research, diagnostics and therapeutics. In general, such nucleic acids may serve as probes that specifically bind to unique sequences of DNA or RNA present in biological samples. This use of nucleic acids may provide for diagnosis of the risk of particular disease states based upon the presence or absence of a particular known gene sequence associated with the disease state. Such nucleotides and nucleic acids (for example, in the form of oligonucleotides) are also used for sequencing lengths or fragments of DNA or RNA from individuals so as to determine their genomic makeup.
Nucleic acid synthesis is typically carried out in a cyclic process that assembles a chain of nucleotides. The nucleotides are added one by one through a series of chemical reactions in which a particular molecule is added to a growing nucleic acid molecule, sometimes via catalysis, until the desired chain is complete. The nucleotides to be added to the chain are typically washed over samples and include blocking molecules that prevent addition of more than one nucleotide at a time in each chain. The blocking molecule is then removed, and the next desired nucleotide may be added in the same way.
Other uses of nucleotides and oligonucleotides include genetic sequencing. Improvements are constantly being made to processes for sequencing nucleic acid segments, which may be supported on a substrate in an array or microarray. In typical DNA sequencing applications nucleotides of the common deoxyribonucleotide types (A, T, C and G) or oligonucleotides containing deoxyribonucleotide monomers are washed over a sample that includes a template DNA to be sequenced and a primer hybridized to the template. A nucleotide or oligonucleotide may bind at a complementary site or sequence of the DNA template that is adjacent to the primer such that the primer is elongated by enzymatically catalyzed addition of the nucleotide or oligonucleotide to the primer. The growing primer is detected in each cycle to determine which nucleotide or oligonucleotide has been incorporated at each site, and the nucleotides are then de-blocked and the cycle repeated. Due to fidelity of the enzymes in specifically adding nucleotides or oligonucleotides that are complementary to the template and rejecting those that are not, the sequence of the template can be determined from the sequence of nucleotides or oligonucleotides added to the primer.
In such processes, nucleotides or oligonucleotides having fluorescent dye markers, blocking molecules, and/or other moieties are typically exposed to the samples in a process fluid. The process fluid also typically contains enzymes that catalyze primer modification. The process fluid is allowed to remain in contact with the sample for a desired time to permit primer modification to take place. The process fluid is then removed in a flush operation, and subsequent processing may occur (e.g., imaging, de-blocking, and so forth). Typically, nucleotides or oligonucleotides are provided in excess amounts to favor high reaction yield. Thus, only a portion of the nucleotides exposed to the samples are consumed and the remainder is flushed away unspent. Similarly, useful enzyme is flushed away after the reaction because under typical conditions the enzyme, being a catalyst, is not consumed.
Current manual and automated systems for both synthesis and sequencing using nucleotides, oligonucleotides and enzymes typically discard these biomolecule reagents contained in the process stream once the stream has been adequately exposed to the sample. However, such molecules can be costly, and the cost of replacing these biomolecule reagents at each cycle can become a significant cost for the overall process.